Evolutionary evidence suggests that Sox3, a member of the high-mobility-group (HMG) family of transcription factors, is an ancestral precursor of Sry and is involved in sex determination similar to Sry. However, there is limited information regarding the SOX3 gene of the black rockfish ( Sebastes schlegeli). In this study, we first isolated SOX3 gene from the gonads of S. schlegeli by homology cloning. The full-length of S. schlegeli SOX3 ( SsSOX3) cDNA was 1386 bp, comprising a 906-bp open reading frame, which encodes a peptide showing 93.6% and 93.9% homology with the Sox3 proteins of Epinephelus coioides and Oryzias latipe, respectively. Comparison of the cDNA sequence of the SsSOX3 gene with the corresponding genomic DNA fragment revealed that the SsSOX3 gene consists of a single exon. Phylogenetic analysis demonstrated the evolutionary relationship of SsSOX3 with other known SOXB1 genes in fish and tetrapods. The promoter region contains binding sites of several transcriptional factors that might participate in the regulation of SsSOX3 expression. Quantitative real-time PCR analysis indicated that SsSOX3 was expressed in all the investigated larval developmental stages from 1 to 35 days after birth and the level of expression gradually decreased as the development proceeded. SsSOX3 exhibited sexually dimorphic expression in adult gonads, with high expression in the ovary but low expression in the testis. In situ hybridization revealed that SsSOX3 was strongly expressed in oocytes and follicular cells of ovaries but slightly expressed in germ cells of testicular tissues. Therefore, this study suggests that SsSOX3 plays an important role in oogenesis and ovary differentiation in S. schlegeli.